The overarching goal of this proposal is to isolate and characterize trans acting factors that regulate stationary phase gene expression in extremely halophilic members of the domain Archaea (i.e., the haloarchaea). Archaeal organisms such as hyperthermophiles, methanogens and extreme halophiles employ a minimalist version of the eucaryal RNA Pol II transcription apparatus. The archaeal apparatus includes TATA box and transcription factor B recognition elements, and homologs to the eucaryal TATA binding protein (TBP), transcription factor lIB (TFB), and the alpha-portion of transcription factor IIE (TFE alpha). Novel to the haloarchaea are multiple copies of the TBPs and TFBs. For example, in Halobacterium species NRC-1, there are 6 tbp genes and seven tfb genes. It has been hypothesized that genes in the haloarchaea may be regulated in part by mixing and matching the TBPs and TFBs in varying combinations. Since the haloarchaea use a Pol II-like transcription system, regulation of stationary phase gene expression will be unlike that seen in the domain Bacteria, where different sigma-factors are employed (e.g., sigma S). To study this question, the stationary phase regulated gene halS8, which encodes the peptide antibiotic microhalocin S8 (HalS8), is used as a model system in a two-pronged approach. First, a wildtype strain (haloarchaeal strain S8a) carrying multiple copies of the halS8 gene on a plasmid is mutagenized, and the survivors screened for a decrease, increase or absence of halocin production. Since all of the halocin S8 genes must be affected in order to produce this phenotype, this approach will produce mutations in genes that encode trans-acting regulatory factors. The genes that have been mutated will be recovered by marker rescue: the mutants will be transformed with a genomic plasmid library and screened for restoration of the wildtype level of halocin production. Second, the multiple copies of the tbp and tfb genes present in strain S8a will be cloned, and then disrupted one-by-one using gene replacement. The effect of the loss of these genes on stationary phase expression will be monitored first using the halocin S8 activity levels, and then second by quantifying halS8 gene transcript levels. Finally, Spanish researchers have shown that microhalocin H7 inhibits the Na+/H+ antiporters in both haloarchaea and mammals. Such inhibitors have been shown to have protective effects against post-ischemic contractile dysfunction. In a dog model, halocin H7 treatment protected the myocardium against the deleterious effects of ischemia and reperfusion by decreasing infarct size and the number of ectopic beats. This finding has implications for reducing injury during organ transplantation. [unreadable] [unreadable] [unreadable]